Protease enzymes for tough cleaning and/or spot and film reduction and compositions incorporating same

ABSTRACT

The present relates to cleaning compositions comprising a protease enzyme which is a carbonyl variant having an amino acid sequence not found in nature, which is derived by replacement of a plurality of amino acid residues of a precursor carbonyl ydrolase with different amino acids.

This application is a 35 U.S.C. §371 application based uponInternational application Ser. No. PCT/IB98/00853, filed Jun. 2 1998,which claims priority to U.S. Provisional Application Ser. No.60/048,550, filed Jun. 4, 1997.

FIELD OF THE INVENTION

The present invention relates to protease enzymes for tough cleaningand/or spot and film reduction in various compositions and methods fortheir use, more particularly to protease enzymes which are carbonylhydrolase variants.

BACKGROUND OF THE INVENTION

Various types of enzymes have long been used in laundry detergents toassist in the removal of certain stains from fabrics. Each class ofenzyme (amylase, protease, etc.) generally catalyzes a differentchemical reaction. For example, protease enzymes are known for theirability to hydrolyze (break down a compound into two or more simplercompounds) other proteins. This ability has been taken advantage ofthrough the incorporation of naturally occurring or engineered proteaseenzymes to laundry detergent compositions.

In recent years the use of enzymes has also been investigated for use inautomatic dishwashing compositions. Unfortunately, many enzymes,especially protease enzymes, do not translate well into the washenvironment. Specifically, thermal stability, pH stability, oxidativestability and substrate specificity need to be optimized to ensuresatisfactory performance.

To optimize the characteristics of the protease enzyme, a change in theamino acid sequence is frequently employed. A change of amino acidsequence may alter the properties of the enzyme to varying degreesdepending upon the location, nature, and/or magnitude of the change inthe amino acid sequence. Several attempts have been made to alter theamino acid sequence of protease enzymes in an attempt to alter theirproperties, with the goal of increasing the efficacy of the protease forcleaning uses such as in the wash environment.

Additionally, consumers interest in automatic dishwashing compositionswhich deliver tough food cleaning is increasing. Baked on dairy productsand eggs have long been difficult to remove via automatic dishwashing.In addition, spotting and filming of glassware is a common problem inautodishwashing. Moreover, consumers now desire less handwashing orpre-washing of dishes and more cleaning ability delivered via theautomatic dishwasher. Accordingly, the need remains for compositionswhich can deliver tough cleaning and/or spot and film reduction cleaningwithout spot/film formation. More particularly, the need remains forautomatic dishwashing compositions which can deliver tough food cleaningand reduced spot/film formation via protease enzymes designed to deliversuch benefits.

BACKGROUND ART

The following documents contain information which may or may not berelevant to the present invention:

WO 95/10615 to Genencor International, Inc.; WO 89/06270 to Novo NordiskA/S; Kirk-Othmer, Encyclopedia of Chemical Technology, 4th. Ed., Vol. 9,Wiley 1994, pages 567-620, titled “Enzyme Applications-Industrial”,Nielsen et al and the references therein. WO 95/10591 and WO 95/10592 tothe Procter & Gamble Company.

SUMMARY OF THE INVENTION

This need is met via the present invention whereby compositions having aprotease enzyme capable of tough food cleaning and reducedspotting/filming is provided. The preferred protease enzyme is acarbonyl hydrolase variant having an amino acid sequence not found innature. The protease is engineered to deliver tough cleaning and/or spotand film reduction and reduced spotting and filming by providing theprotease with trypsin-like specificity. Thus, the protease is highlyeffective on dairy soils such as milk and cheese and on egg yolk soilsand significantly reduces the spotting and filming such soils may causein the automatic dishwashing process. The protease is derived byreplacement of a plurality of amino acid residues of a precursorcarbonyl hydrolase with different amino acids. Furthermore, thepreferred protease is engineered to have a higher level of bleachstability. In addition, the enzymes of the present invention may provideimproved soil removal in laundry applications as well.

According to a first embodiment of the present invention, a cleaningcomposition is provided. The cleaning composition comprises:

(a) an effective amount of a protease enzyme which is a carbonylhydrolase variant having an amino acid sequence not found in nature,which is derived by replacement of a plurality of amino acid residues ofa precursor carbonyl hydrolase with different amino acids, wherein saidplurality of amino acid residues replaced in the precursor enzymecorrespond to position +210 in combination with one or more of thefollowing residues: +33, +62, +67, +76, +100, +101, +103, +104, +107,+128, +129, +130, +132, +135, +156, +158, +164, +166, +167, +170, +209,+215, +217, +218 and +222, where the numbered positions correspond tonaturally-occurring subtilisin from Bacillus amyloliquefaciens or toequivalent amino acid residues in other carbonyl hydrolases orsubtilisins (such as Bacillus lentus subtilisin); and

(b) one or more cleaning adjunct materials compatible with the proteaseenzyme.

Most preferably, the protease is derived by replacement of a pluralityof amino acid residues of a precursor carbonyl hydrolase with differentamino acids, wherein the plurality of amino acid residues replaced inthe precursor enzyme correspond to position +210 in combination with oneor more of the following residues: +76, +103, +104, +156, +166, and+217, +222, and most preferably, the protease is derived from replacingamino acid residues at positions +210, +76, +103, +104, +156, and +166.

The present invention also relates to methods for cleaning items in needof cleaning by contacting the item with a protease enzyme which is acarbonyl hydrolase variant as described herein. The invention thereforeencompasses a method for cleaning fabrics comprising contacting,preferably with agitation, the fabrics with an aqueous liquor containingthe protease enzyme. The method can be carried out at temperatures belowabout 60° C. but, of course, is quite effective at laundry temperaturesup to the boil. The present invention also relates to a method forcleaning dishes by contacting a dish in need of cleaning with a proteaseenzyme as described herein. The present invention methods also includemethods for personal cleansing, the methods comprising contacting thepart of the human or lower animal body in need of cleaning with aprotease enzyme as described herein.

Accordingly, it is an object of the present invention to provide acleaning composition having a protease enzyme capable of tough cleaning,and/or spot and film reduction particularly an automatic dishwashingcomposition having tough soil or food cleaning. It is further an objectof the present invention to provide methods for fabric, dish andpersonal cleansing via the use of the protease enzymes of the presentinvention. These, and other, objects, features and advantages will beclear from the following detailed description, the attached drawings andthe appended claims.

All percentages, ratios and proportions herein are on a weight basisunless otherwise indicated. All documents cited herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict the DNA and amino acid sequence of subtilisinfrom Bacillus lentus (Seq. ID No. 6 and 7). The mature subtilisinprotein is coded by the codons beginning at the codon GCG (334-336)corresponding to Ala.

FIGS. 2A and 2B depict the amino acid sequence of four subtilisins. Thetop line represents the amino acid sequence of subtilisin from Bacillusamyloliquefaciens subtilisin (also sometimes referred to as subtilisinBPN′) (Seq. ID No. 2). The second line depicts the amino acid sequenceof subtilisin from Bacillus subtilis (Seq. ID No. 3). The third linedepicts the amino acid sequence of subtilisin from B. licheniformis(Seq. ID No. 4). The fourth line depicts the amino acid sequence ofsubtilisin from Bacillus lentus (also referred to as subtilisin 309 inPCT WO 89/06276) (Seq. ID No. 5). The symbol * denotes the absence ofspecific amino acid residues as compared to subtilisin BPN′.

FIGS. 3A-C depict the DNA and amino acid sequence for Bacillusamyloliquefaciens subtilisin and a partial restriction map of this gene(Seq. ID No. 1).

FIG. 4 depicts the conserved amino acid residues among subtilisins fromBacillus amyloliquefaciens (BPN′) and Bacillus lentus (wild-type).

FIGS. 5A and 5B depict the DNA and amino acid sequence of a preferredembodiment of the invention (P210I/S156E/S166D/N76D/S103A/V104I) (Seq.ID No. 8 and 9). The DNA in this figure has been modified by the methodsdescribed to encode aspartate at positions 76 and 166, glutamate atposition 156, alanine at position 103 and isoleucine at positions 210and 104. The mature subtilisin variant protein is coded by the codonsbeginning at the codon GCG (334-336) corresponding to ala.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Protease Enzymes—The invention includes protease enzymes which arenon-naturally-occurring carbonyl hydrolase variants having a differentproteolytic activity, stability, substrate specificity, pH profileand/or performance characteristic as compared to the precursor carbonylhydrolase from which the amino acid sequence of the variant is derived.As stated earlier, the protease enzymes are designed to havetrypsin-like specificity and preferably also be bleach stable. Theprecursor carbonyl hydrolase may be a naturally-occurring carbonylhydrolase or recombinant hydrolase. Specifically, such carbonylhydrolase variants have an amino acid sequence not found in nature,which is derived by replacement of a plurality of amino acid residues ofa precursor carbonyl hydrolase with different amino acids. The pluralityof amino acid residues of the precursor enzyme correspond to position+210 in combination with one or more of the following residues: +33,+62, +67, +76, +100, +101, +103, +104, +107, +128, +129, +130, +132,+135, +156, +158, +164, +166, +167, +170, +209, +215, +217, +218, and+222, where the numbered position corresponds to naturally-occurringsubtilisin from Bacillus amyloliquefaciens or to equivalent amino acidresidues in other carbonyl hydrolases or subtilisins, such as Bacilluslentus subtilisin.

The carbonyl hydrolase variants which are protease enzymes useful in thepresent invention compositions comprise replacement of amino acidresidue +210 in combination with one or more additional modifications.While any combination of the above listed amino acid substitutions maybe employed, the preferred variant protease enzymes useful for thepresent invention comprise the substitution, deletion or insertion ofamino acid residues in the following combinations: 210/156; 210/166;210/76; 210/103; 210/104; 210/217; 210/156/166; 210/156/217;210/166/217; 210/76/156; 210/76/166; 210/76/217; 210/76/156/166;210/76/156/217; 210/76/166/217; 210/76/103/156; 210/76/103/166;210/76/103/217; 210/76/104/156; 210/76/104/166; 210/76/104/217;210/76/103/104/156; 210/76/103/104/166; 210/76/103/104/217;210/76/103/104/156/166; 210/76/103/104/156/217; 210/76/103/104/166/217and/or 210/76/103/104/156/166/217; 210/76/103/104/166/222;210/67/76/103/104/166/222; 210/67/76/103/104/166/218/222. Mostpreferably the variant enzymes useful for the present invention comprisethe substitution, deletion or insertion of an amino acid residue in thefollowing combination of residues: 210/156; 210/166; 210/217;210/156/166; 210/156/217; 210/166/217; 210/76/156/166;210/76/103/156/166 and 210/76/103/104/156/166 of B. lentus subtilisinwith 210/76/103/104/156/166 being the most preferred.

Variant DNA sequences encoding such carbonyl hydrolase or subtilisinvariants are derived from a precursor DNA sequence which encodes anaturally-occurring or recombinant precursor enzyme. The variant DNAsequences are derived by modifying the precursor DNA sequence to encodethe substitution of one or more specific amino acid residues encoded bythe precursor DNA sequence corresponding to positions +210, +33, +62,+67, +76, +100, +101, +103, +104, +107, +128, +129, +130, +132, +135,+156, +158, +164, +166, +167, +170, +209, +215, +217, +218, and +222 inBacillus lentus or any combination thereof. Although the amino acidresidues identified for modification herein are identified according tothe numbering applicable to B. amyloliquefaciens (which has become theconventional method for identifying residue positions in allsubtilisins), the preferred precursor DNA sequence useful for thepresent invention is the DNA sequence of Bacillus lentus as shown inFIG. 1.

These variant DNA sequences encode the insertion or substitution of theamino acid residue +210 in combination with one or more additionalmodification. Preferably the variant DNA sequences encode thesubstitution or insertion of amino acid residues in the followingcombinations: 210/156; 210/166; 210/76; 210/103; 210/104; 210/217;210/156/166; 210/156/217; 210/166/217; 210/76/156; 210/76/166;210/76/217; 210/76/156/166; 210/76/156/217; 210/76/166/217;210/76/103/156; 210/76/103/166; 210/76/103/217; 210/76/104/156;210/76/104/166; 210/76/104/217; 210/76/103/104/156; 210/76/103/104/166;210/76/103/104/217; 210/76/103/104/156/166; 210/76/103/104/156/217;210/76/103/104/166/217 and/or 210/76/103/104/156/166/217;210/76/103/104/166/222; 210/67/76/103/104/166/222;210/67/76/103/104/166/218/222. Most preferably the variant DNA sequencesencode for the modification of the following combinations of residues:210/156; 210/166; 210/217; 210/156/166; 210/156/217; 210/166/217;210/76/156/166; 210/76/103/156/166 and 210/76/103/104/156/166. Theserecombinant DNA sequences encode carbonyl hydrolase variants having anovel amino acid sequence and, in general, at least one property whichis substantially different from the same property of the enzyme encodedby the precursor carbonyl hydrolase DNA sequence. Such propertiesinclude proteolytic activity, substrate specificity, stability, alteredpH profile and/or enhanced performance characteristics.

The protease enzymes useful herein encompass the substitution of any ofthe nineteen naturally occurring L-amino acids at the designated aminoacid residue positions. Such substitutions can be made in any precursorsubtilisin (procaryotic, eucaryotic, mammalian, etc.). Throughout thisapplication reference is made to various amino acids by way of commonone- and three-letter codes. Such codes are identified in Dale, M. W.(1989), Molecular Genetics of Bacteria, John Wiley & Sons, Ltd.,Appendix B.

Preferably, the substitution to be made at each of the identified aminoacid residue positions include but are not limited to substitutions atposition +210 including I, V, L, and A, substitutions at positions +33,+62, +76, +100, +101, +103, +104, +107, +128, +129, +130, +132, +135,+156, +158, +164, +166, +167, +170, +209, +215, +217, and +218 of D orE, substitutions at position 76 including D, H, E, G, F, K, P and N;substitutions at position 103 including Q, T, D, E, Y, K, G, R and S;and substitutions at position 104 including S, Y, I, L, M, A, W, D, T, Gand V; and substitutions at position 222 including S, C, A. Thespecifically preferred amino acid(s) to be substituted at each suchposition are designated below in Table 1. Although specific amino acidsare shown in Table I, it should be understood that any amino acid may besubstituted at the identified residues.

TABLE I Amino Acid Preferred Amino Acid to Residue beSubstituted/Inserted +210 I, V, L, A +33, +62, +100, +101, +107 D,E+128, +129, +130, +135 +156, +158, +164, +166 +167, +170, +209, +215+217 and +218 +76 D,H +103 A,Q,T,D,B,Y,K,G,R +104 I,Y,S,L,A,T,G +222S,C,A

Carbonyl hydrolases are protease enzymes which hydrolyze compoundscontaining

bonds in which X is oxygen or nitrogen. They include naturally-occurringcarbonyl hydrolases and recombinant carbonyl hydrolases.Naturally-occurring carbonyl hydrolases principally include hydrolases,e.g., peptide hydrolases such as subtilisins or metalloproteases.Peptide hydrolases include α-aminoacylpeptide hydrolase, peptidylaminoacid hydrolase, acylamino hydrolase, serine carboxypeptidase,metallocarboxypeptidase, thiol proteinase, carboxylproteinase andmetalloproteinase. Serine, metallo, thiol and acid protease's areincluded, as well as endo and exo-proteases.

“Recombinant carbonyl hydrolase” refers to a carbonyl hydrolase in whichthe DNA sequence encoding the naturally-occurring carbonyl hydrolase ismodified to produce a mutant DNA sequence which encodes thesubstitution, insertion or deletion of one or more amino acids in thecarbonyl hydrolase amino acid sequence. Suitable modification methodsare disclosed herein, and in U.S. Pat. Nos. 4,760,025, 5,204,015 and5,185,258, the disclosure of which are incorporated herein by reference.

Subtilisins are bacterial or fungal carbonyl hydrolases which generallyact to cleave peptide bonds of proteins or peptides. As used herein,“subtilisin” means a naturally-occurring subtilisin or a recombinantsubtilisin. A series of naturally-occurring subtilisins is known to beproduced and often secreted by various microbial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus, is aspartate-histidine-serine. In thechymotrypsin related proteases the relative order, however, ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.Examples include but are not limited to the subtilisins identified inFIG. 2 herein.

“Recombinant subtilisin” refers to a subtilisin in which the DNAsequence encoding the subtilisin is modified to produce a variant (ormutant) DNA sequence which encodes the substitution, deletion orinsertion of one or more amino acids in the naturally-occurringsubtilisin amino acid sequence. Suitable methods to produce suchmodification and which may be combined with those disclosed herein,include those disclosed in U.S. Pat. Nos. 4,760,025, 5,204,015 and5,185,258.

“Non-human carbonyl hydrolases” and the DNA encoding them may beobtained from many procaryotic and eucaryotic organisms. Suitableexamples of procaryotic organisms include gram negative organisms suchas E. coli or Pseudomonas and gram positive bacteria such as Micrococcusor Bacillus. Examples of eucaryotic organisms from which carbonylhydrolase and their genes may be obtained include yeast such asSaccharomyces cerevisiae, fungi such as Aspergillus sp. and non-humanmammalian sources such as, for example, bovine sp. from which the geneencoding the carbonyl hydrolase chymosin can be obtained. As withsubtilisins, a series of carbonyl hydrolases can be obtained fromvarious related species which have amino acid sequences which are notentirely homologous between the members of that series but whichnevertheless exhibit the same or similar type of biological activity.Thus, non-human carbonyl hydrolase as used herein has a functionaldefinition which refers to carbonyl hydrolases which are associated,directly or indirectly, with procaryotic and eucaryotic sources.

A “carbonyl hydrolase variant” has an amino acid sequence which isderived from the amino acid sequence of a “precursor carbonylhydrolase.” The precursor carbonyl hydrolases includenaturally-occurring carbonyl hydrolases and recombinant carbonylhydrolases. The amino acid sequence of the carbonyl hydrolase variant is“derived” from the precursor hydrolase amino acid sequence by thesubstitution, deletion or insertion of one or more amino acids of theprecursor amino acid sequence. Such modification is of the “precursorDNA sequence” which encodes the amino acid sequence of the precursorcarbonyl hydrolase rather than manipulation of the precursor carbonylhydrolase enzyme per se. Suitable methods for such manipulation of theprecursor DNA sequence include methods disclosed herein and in U.S. Pat.No. 4,760,025.

Specific residues corresponding to position +210 in combination with oneor more of the following positions +33, +62, +67, +76, +100, +101, +103,+104, +107, +128, +129, +130, +132, +135, +156, +158, +164, +166, +167,+170, +209, +215, +217, +218 and +222 of Bacillus lentus subtilisin areidentified herein for mutation. Preferably the modified residues areselected from the following combinations: 210/156; 210/166; 210/76;210/103; 210/104; 210/217; 210/156/166; 210/156/217; 210/166/217;210/76/156; 210/76/166; 210/76/217; 210/76/156/166; 210/76/156/217;210/76/166/217; 210/76/103/156; 210/76/103/166; 210/76/103/217;210/76/104/156; 210/76/104/166; 210/76/104/217; 210/76/103/104/156;210/76/103/104/166; 210/76/103/104/217; 210/76/103/104/156/166;210/76/103/104/156/217; 210/76/103/104/166/217 and/or210/76/103/104/156/166/217 with preferred combinations being: 210/156;210/166; 210/217; 210/156/166; 210/156/217; 210/166/217; 210/76/156/166;210/76/103/156/166 and 210/76/103/104/156/166. These amino acid positionnumbers refer to those assigned to the mature Bacillus amyloliquefacienssubtilisin sequence presented in FIG. 3. The protease enzymes useful inthe present invention, however, are not limited to the mutation of thisparticular subtilisin but extends to precursor carbonyl hydrolasescontaining amino acid residues at positions which are “equivalent” tothe particular identified residues in Bacillus amyloliquefacienssubtilisin. Preferably, the precursor subtilisin is Bacillus lentussubtilisin and the substitutions, deletions or insertions are made atthe equivalent amino acid residue in B. lentus corresponding to thoselisted above.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalentto a residue of Bacillus amyloliquefaciens subtilisin if it is eitherhomologous (i.e., corresponding in position in either primary ortertiary structure) or analogous to a specific residue or portion ofthat residue in Bacillus amyloliquefaciens subtilisin (i.e., having thesame or similar functional capacity to combine, react, or interactchemically).

In order to establish homology to primary structure, the amino acidsequence of a precursor carbonyl hydrolase is directly compared to theBacillus amyloliquefaciens subtilisin primary sequence and particularlyto a set of residues known to be invariant in subtilisins for whichsequence is known. FIG. 4 herein shows the conserved residues as betweenamyloliquefaciens subtilisin and B. lentus subtilisin. After aligningthe conserved residues, allowing for necessary insertions and deletionsin order to maintain alignment (i.e., avoiding the elimination ofconserved residues through arbitrary deletion and insertion), theresidues equivalent to particular amino acids in the primary sequence ofBacillus amyloliquefaciens subtilisin are defined. Alignment ofconserved residues preferably should conserve 100% of such residues.However, alignment of greater than 75% or as little as 50% of conservedresidues is also adequate to define equivalent residues. Conservation ofthe catalytic triad, Asp32/His64/Ser221 should be maintained.

For example, in FIG. 2 the amino acid sequence of subtilisin fromBacillus amyloliquefaciens, Bacillus subtilis, Bacillus licheniformis(carlsbergensis) and Bacillus lentus are aligned to provide the maximumamount of homology between amino acid sequences. A comparison of thesesequences shows that there are a number of conserved residues containedin each sequence. These conserved residues (as between BPN′ and B.lentus) are identified in FIG. 4.

These conserved residues, thus, may be used to define the correspondingequivalent amino acid residues of Bacillus amyloliquefaciens subtilisinin other carbonyl hydrolases such as subtilisin from Bacillus lentus(PCT Publication No. W089/06279 published Jul. 13, 1989) and thepreferred subtilisin precursor enzyme herein. These particular aminoacid sequences are aligned in FIGS. 2A and 2B with the sequence ofBacillus amyloliquefaciens subtilisin to produce the maximum homology ofconserved residues. As can be seen, there are a number of deletions inthe sequence of Bacillus lentus as compared to Bacillusamyloliquefaciens subtilisin. Thus, for example, the equivalent aminoacid for Vall65 in Bacillus amyloliquefaciens subtilisin in the othersubtilisins is isoleucine for B. lentus and B. licheniformis.

Thus, for example, the amino acid at position +210 is proline (P) inboth B. amyloliquefaciens and B. lentus subtilisins. In the preferredsubtilisin variant useful in the invention, however, the amino acidequivalent to +210 in Bacillus amyloliquefaciens subtilisin issubstituted with isoleucine (I). A comparison of the preferred aminoacid residues identified herein for substitution versus the preferredsubstitution for each such position is provided in Table II.

TABLE II +210 +156 +166 +217 +76 +103 +104 B. amyloliquefaciens P E G YN Q Y (wild-type) B. lentus (wild-type) P S S L N S V Most Preferred IE/D E/D E/D D A I/Y Substitution

Equivalent residues may also be defined by determining homology at thelevel of tertiary structure for a precursor carbonyl hydrolase whosetertiary structure has been determined by x-ray crystallography.Equivalent residues are defined as those for which the atomiccoordinates of two or more of the main chain atoms of a particular aminoacid residue of the precursor carbonyl hydrolase and Bacillusamyloliquefaciens subtilisin (N on N, CA on CA, C on C and O on O) arewithin 0.13 nm and preferably 0.1 nm after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the carbonyl hydrolase in question to the Bacillus amyloliquefacienssubtilisin. The best model is the crystallographic model giving thelowest R factor for experimental diffraction data at the highestresolution available.${R\quad {factor}} = {\frac{{\Sigma_{h}{{{Fo}(h)}}} - {{{Fc}(h)}}}{\quad}\Sigma_{h}{{{Fo}(h)}}}$

Equivalent residues which are functionally analogous to a specificresidue of Bacillus amyloliquefaciens subtilisin are defined as thoseamino acids of the precursor carbonyl hydrolases which may adopt aconformation such that they either alter, modify or contribute toprotein structure, substrate binding or catalysis in a manner definedand attributed to a specific residue of the Bacillus amyloliquefacienssubtilisin. Further, they are those residues of the precursor carbonylhydrolase (for which a tertiary structure has been obtained by x-raycrystallography) which occupy an analogous position to the extent that,although the main chain atoms of the given residue may not satisfy thecriteria of equivalence on the basis of occupying a homologous position,the atomic coordinates of at least two of the side chain atoms of theresidue lie with 0.13 nm of the corresponding side chain atoms ofBacillus amyloliquefaciens subtilisin. The coordinates of the threedimensional structure of Bacillus amyloliquefaciens subtilisin are setforth in EPO Publication No. 0 251 446 (equivalent to U.S. patentapplication Ser. No. 07/898,382, the disclosure of which is incorporatedherein by reference) and can be used as outlined above to determineequivalent residues on the level of tertiary structure.

Some of the residues identified for substitution, insertion or deletionare conserved residues whereas others are not. In the case of residueswhich are not conserved, the replacement of one or more amino acids islimited to substitutions which produce a variant which has an amino acidsequence that does not correspond to one found in nature. In the case ofconserved residues, such replacements should not result in anaturally-occurring sequence. The carbonyl hydrolase variants useful inthe present invention include the mature forms of carbonyl hydrolasevariants, as well as the pro- and prepro-forms of such hydrolasevariants. The prepro-forms are the preferred construction since thisfacilitates the expression, secretion and maturation of the carbonylhydrolase variants.

Methods and procedures for making the enzymes according to the presentinvention are known and are disclosed in PCT Publication No. WO 95/10615the disclosure of which is herein incorporated by reference.

The enzymes of the present invention have trypsin-like specificity. Thatis, the enzymes of the present invention hydrolyze proteins bypreferentially cleaving the peptide bonds of charged amino acidresidues, more specifically residues such as arginine and lysine, ratherthan preferentially cleaving the peptide bonds of hydrophobic amino acidresidues, more specifically phenylalanine, tryptophan and tyrosine.Enzymes having the latter profile have a chymotrypsin-like specificity.Substrate specificity as discussed above is illustrated by the action ofthe enzyme on two synthetic substrates. Protease's having trypsin-likespecificity hydrolyze the synthetic substrate bVGR-pNA preferentiallyover the synthetic substrate sucAAPF-pNA. Chymotrypsin-like proteaseenzymes, in contrast, hydrolyze the latter much faster than the former.For the purposes of the present invention the following procedure wasemployed to define the trypsin-like specificity of the protease enzymesof the present invention:

A fixed amount of a glycine buffer at a pH of 10 and a temperature of 25° C. is added to a standard 10 ml test tube. 0.5 ppm of the activeenzyme to be tested is added to the test tube. Approximately, 1.25 mg ofthe synthetic substrate per mL of buffer solution is added to the testtube. The mixture is allowed to incubate for 15 minutes at 25° C. Uponcompletion of the incubation period, an enzyme inhibitor, PMSF, is addedto the mixture at a level of 0.5 mg per mL of buffer solution. Theabsorbency or OD value of the mixture is read at a 410 nm wavelength.The absorbence then indicates the activity of the enzyme on thesynthetic substrate. The greater the absorbence, the higher the level ofactivity against that substrate.

To then determine the specificity of an individual enzyme, theabsorbence on the two synthetic substrate proteins may be converted intoa specificity ratio. For the purposes of the present invention, theratio is determined by the formula specificity of:

[activity on sAAPF-pNA]/[activity on bVGR-pNA]

An enzyme having a ratio of less than about 10, more preferably lessthan about 5 and most preferably less than about 2.5 may then beconsidered to demonstrate trypsin-like activity.

Cleaning Adjunct Materials—The cleaning compositions of the presentinvention also comprise, in addition to the protease enzyme describedhereinbefore, one or more cleaning adjunct materials compatible with theprotease enzyme. The term “cleaning adjunct materials”, as used herein,means any liquid, solid or gaseous material selected for the particulartype of cleaning composition desired and the form of the product (e.g.,liquid; granule; spray composition), which materials are also compatiblewith the protease enzyme used in the composition. The specific selectionof cleaning adjunct materials are readily made by considering thesurface, item or fabric to be cleaned, and the desired form of thecomposition for the cleaning conditions during use (e.g., through thewash detergent use). The term “compatible”, as used herein, means thecleaning composition materials do not reduce the proteolytic activity ofthe protease enzyme to such an extent that the protease is not effectiveas desired during normal use situations. Specific cleaning compositionmaterials are exemplified in detail hereinafter.

An effective amount of one or more protease enzymes described above areincluded in compositions useful for cleaning a variety of surfaces inneed of proteinaceous stain removal. Such cleaning compositions includedetergent compositions for cleaning hard surfaces, unlimited in form(e.g., liquid and granular); detergent compositions for cleaningfabrics, unlimited in form (e.g., granular, liquid and barformulations); dishwashing compositions (unlimited in form and includingboth granular and liquid automatic dishwashing); oral cleaningcompositions, unlimited in form (e.g., dentifrice, toothpaste andmouthwash formulations); and denture cleaning compositions, unlimited inform (e.g., liquid, tablet). As used herein, “effective amount ofprotease enzyme” refers to the quantity of protease enzyme describedhereinbefore necessary to achieve the enzymatic activity necessary inthe specific cleaning composition. Such effective amounts are readilyascertained by one of ordinary skill in the art and is based on manyfactors, such as the particular enzyme variant used, the cleaningapplication, the specific composition of the cleaning composition, andwhether a liquid or dry (e.g., granular, bar) composition is required,and the like.

Preferably the cleaning compositions of the present invention comprisefrom about 0.0001% to about 10% of one or more protease enzymes, morepreferably from about 0.001% to about 1%, more preferably still fromabout 0.001% to about 0. 1%. Also preferably the protease enzyme ispresent in the compositions in an amount sufficient to provide a ratioof mg of active protease per 100 grams of composition to ppm theoreticalAvailable O₂ (“AvO₂”) from any peroxyacid in the wash liquor, referredto herein as the Enzyme to Bleach ratio (E/B ratio), ranging from about1:1 to about 20:1. Several examples of various cleaning compositionswherein the protease enzymes may be employed are discussed in furtherdetail below. Also, the compositions of the present invention mayinclude from about 1% to about 99.9% by weight of the composition of theadjunct materials.

Optional Detersive Enzymes—The detergent compositions herein may alsooptionally contain one or more types of detergent enzymes. Such enzymescan include proteases, amylases, cellulases and lipases. Such materialsare known in the art and are commercially available under suchtrademarks as. They may be incorporated into the non-aqueous liquiddetergent compositions herein in the form of suspensions, “marumes” or“prills”. Another suitable type of enzyme comprises those in the form ofslurries of enzymes in nonionic surfactants, e.g., the enzymes marketedby Novo Nordisk under the tradename “SL” or the microencapsulatedenzymes marketed by Novo Nordisk under the tradename “LDP.” Suitableenzymes and levels of use are described in U.S. Pat. No. 5,576,282,5,705,464 and 5,710,115.

Enzymes added to the compositions herein in the form of conventionalenzyme prills are especially preferred for use herein. Such prills willgenerally range in size from about 100 to 1,000 microns, more preferablyfrom about 200 to 800 microns and will be suspended throughout thenon-aqueous liquid phase of the composition. Prills in the compositionsof the present invention have been found, in comparison with otherenzyme forms, to exhibit especially desirable enzyme stability in termsof retention of enzymatic activity over time. Thus, compositions whichutilize enzyme prills need not contain conventional enzyme stabilizingsuch as must frequently be used when enzymes are incorporated intoaqueous liquid detergents.

“Detersive enzyme”, as used herein, means any enzyme having a cleaning,stain removing or otherwise beneficial effect in a laundry, hard surfacecleaning or personal care detergent composition. Preferred detersiveenzymes are hydrolases such as proteases, amylases and lipases.Preferred enzymes for laundry purposes include, but are not limited to,proteases, cellulases, lipases and peroxidases. Highly preferred forautomatic dishwashing are amylases and/or proteases, including bothcurrent commercially available types and improved types which, thoughmore and more bleach compatible though successive improvements, have aremaining degree of bleach deactivation susceptibility.

Examples of suitable enzymes include, but are not limited to,hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases,phospholipases, esterases, cutinases, pectinases, keratanases,reductases, oxidases, phenoloxidases, lipoxygenases, ligninases,pullulanases, tannases, pentosanases, malanases, β-glucanases,arabinosidases, hyaluronidase, chondroitinase, laccase, and knownamylases, or mixtures thereof.

Examples of such suitable enzymes are disclosed in U.S. Pat. Nos.5,705,464, 5,710,115, 5,576,282, 5,728,671 and 5,707,950

Particularly useful proteases are described in PCT publications: WO95/30010 published Nov. 9, 1995 by The Procter & Gamble Company; WO95/30011 published Nov. 9, 1995 by The Procter & Gamble Company; and WO95/29979 published Nov. 9, 1995 by The Procter & Gamble Company.Suitable proteases are commercially available as ESPEPASE®, ALCALASE®,DURAZYM® and SAVINASE® all from Novo Nordisk A/S of Denmark, and asMAXATASE®, MAXACAL®, PROPERASE®, and MAXAPEM® all from Gist-Brocades ofThe Netherlands.

In addition to the peroxidase enzymes disclosed in U.S. Pat. Nos.5,705,464, 5,710,115, 5,576,282, 5,728,671 and 5,707,950, other suitableperoxidase enzymes are disclosed in European Patent application EP No.96870013.8, filed Feb. 20, 1996. Also suitable is the laccase enzyme.

Preferred enhancers are substituted phenthiazine and phenoxasine10-Phenothiazinepropionicacid (PPT), 10-ethylphenothiazine-4-carboxylicacid (EPC), 10-phenoxazinepropionic acid (POP) and 10-methylphenoxazine(described in WO 94/12621) and substitued syringates (C3-C5 substituedalkyl syringates) and phenols. Sodium percarbonate or perborate arepreferred sources of hydrogen peroxide.

Said peroxidases are normally incorporated in the detergent compositionat levels from 0.0001% to 2% of active enzyme by weight of the detergentcomposition.

Other preferred enzymes that can be included in the detergentcompositions of the present invention include lipases. Suitable lipaseenzymes for detergent usage include those produced by microorganisms ofthe Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, asdisclosed in British Patent 1,372,034. Suitable lipases include thosewhich show a positive immunological cross-reaction with the antibody ofthe lipase, produced by the microorganism Pseudomonas fluorescent IAM1057. This lipase is available from Amano Pharmaceutical Co. Ltd.,Nagoya, Japan, under the trade name Lipase P “Amano,” hereinafterreferred to as “Amano-P”. Other suitable commercial lipases includeAmano-CES, lipases ex Chromobacter viscosum, e.g. Chromobacter viscosumvar. lipolyticum NRRLB 3673 from Toyo Jozo Co., Tagata, Japan;Chromobacter viscosum lipases from U.S. Biochemical Corp., U.S.A. andDisoynth Co., The Netherlands, and lipases ex Pseudomonas gladioli.Especially suitable lipases are lipases such as M1 LIPASE® andLIPOMAX®(Gist-Brocades) and LIPOLASE® and LIPOLASE ULTRA® (Novo) whichhave found to be very effective when used in combination with thecompositions of the present invention.

Also suitable are cutinases [EC 3.1.1.50] which can be considered as aspecial kind of lipase, namely lipases which do not require interfacialactivation. Addition of cutinases to detergent compositions have beendescribed in e.g. WO 88/09367 (Genencor).

In addition to the above referenced lipases, phospholipases may beincorporated into the detergent compositions of the present invention.Nonlimiting examples of suitable phospholipases included: EC 3.1.1.32Phospholipase A1; EC 3.1.1.4 Phospholipase A2; EC 3.1.1.5 Lysopholipase;EC 3.1.4.3 Phospholipase C; EC 3.1.4.4. Phospolipase D. Commerciallyavailable phospholipases include LECITASE® from Novo Nordisk A/S ofDenmark and Phospholipase A2 from Sigma. When phospolipases are includedin the compositions of the present invention, it is preferred thatamylases are also included. Without desiring to be bound by theory, itis believed that the combined action of the phospholipase and amylaseprovide substantive stain removal, especially on greasy/oily, starchyand highly colored stains and soils. Preferably, the phospholipase andamylase, when present, are incorporated into the compositions of thepresent invention at a pure enzyme weight ratio between 4500:1 and 1:5,more preferably between 50:1 and 1:1.

Known amylases (α and/or β) can be included for removal ofcarbohydrate-based stains. WO 94/02597, Novo Nordisk A/S published Feb.3, 1994, describes cleaning compositions which incorporate mutantamylases. See also WO94/18314, Genencor, published Aug. 18, 1994 andWO95/10603, Novo Nordisk A/S, published Apr. 20, 1995. Other amylasesknown for use in detergent compositions include both α- and β-amylases.α-Amylases are known in the art and include those disclosed in U.S. Pat.No. 5,003,257; EP 252,666; WO 91/00353; FR 2,676,456; EP 285,123; EP525,610; EP 368,341; and British Patent Specification No. 1,296,839(Novo). Other suitable amylase are stability-enhanced amylases includingPURAFACT OX AM® described in WO 94/18314, published Aug. 18, 1994 andWO96/05295, Genencor, published Feb. 22, 1996 and amylase variants fromNovo Nordisk A/S, disclosed in WO 95/10603, published April 1995.

Examples of commercial α-amylases products are TERMAMYL®, BAN®,FUNGAMYL® and DURAMYL®, all available from Novo Nordisk A/S Denmark.WO95/26397 describes other suitable amylases: α-amylases characterizedby having a specific activity at least 25% higher than the specificactivity of TERMAMYL® at a temperature range of 25° C. to 55° C. and ata pH value in the range of 8 to 10, measured by the Phadebas® α-amylaseactivity assay. Other amylolytic enzymes with improved properties withrespect to the activity level and the combination of thermostability anda higher activity level are described in WO95/35382.

The above-mentioned enzymes may be of any suitable origin, such asvegetable, animal, bacterial, fungal and yeast origin. Purified ornon-purified forms of these enzymes may be used. Also included bydefinition, are mutants of native enzymes. Mutants can be obtained e.g.by protein and/or genetic engineering, chemical and/or physicalmodifications of native enzymes. Common practice as well is theexpression of the enzyme via host organisms in which the geneticmaterial responsible for the production of the enzyme has been cloned.

Said enzymes are normally incorporated in the detergent composition atlevels from 0.0001% to 2% of active enzyme by weight of the detergentcomposition. The enzymes can be added as separate single ingredients(prills, granulates, stabilized liquids, etc. containing one enzyme ) oras mixtures of two or more enzymes ( e.g. cogranulates).

Other suitable detergent ingredients that can be added are enzymeoxidation scavengers. Examples of such enzyme oxidation scavengers areethoxylated tetraethylene polyamines.

A range of enzyme materials and means for their incorporation intosynthetic detergent compositions is also disclosed in WO 9307263 and WO9307260 to Genencor International, WO 8908694 to Novo, and U.S. Pat. No.3,553,139, Jan. 5, 1971 to McCarty et al. Enzymes are further disclosedin U.S. Pat. No. 4,101,457, Place et al, Jul. 18, 1978, and in U.S. Pat.No. 4,507,219, Hughes, Mar. 26, 1985. Enzyme materials useful for liquiddetergent formulations, and their incorporation into such formulations,are disclosed in U.S. Pat. No. 4,261,868, Hora et al, Apr. 14, 1981.

Enzyme Stabilizers—Enzymes for use in detergents can be stabilized byvarious techniques. Enzyme stabilization techniques are disclosed andexemplified in U.S. Pat. No. 3,600,319, Aug. 17, 1971, Gedge et al, EP199,405 and EP 200,586, Oct. 29, 1986, Venegas. Enzyme stabilizationsystems are also described, for example, in U.S. Pat. No. 3,519,570. Auseful Bacillus, sp. AC13 giving proteases, xylanases and cellulases, isdescribed in WO 9401532 to Novo. The enzymes employed herein can bestabilized by the presence of water-soluble sources of calcium and/ormagnesium ions in the finished compositions which provide such ions tothe enzymes. Suitable enzyme stabilizers and levels of use are describedin U.S. Pat. Nos. 5,705,464, 5,710,115 and 5,576,282.

Chelating Agents—The detergent compositions herein may also optionallycontain a chelating agent which serves to chelate metal ions, e.g., ironand/or manganese, within the non-aqueous detergent compositions herein.Such chelating agents thus serve to form complexes with metal impuritiesin the composition which would otherwise tend to deactivate compositioncomponents such as the peroxygen bleaching agent. Useful chelatingagents can include amino carboxylates, phosphonates, amino phosphonates,polyfunctionally-substituted aromatic chelating agents and mixturesthereof. Further examples of suitable chelating agents and levels of useare described in U.S. Pat. Nos. 5,705,464, 5,710,115 and 5,576,282.

Organic Builders—The compositions herein also optionally, butpreferably, contain up to about 50%, more preferably from about 1% toabout 40%, even more preferably from about 5% to about 30%, by weight ofa detergent builder material. Lower or higher levels of builder,however, are not meant to be excluded. Detergent builders can optionallybe included in the compositions herein to assist in controlling mineralhardness. Inorganic as well as organic builders can be used. Buildersare typically used in fabric laundering compositions to assist in theremoval of particulate soils. Suitable detergent builders are describedin U.S. Pat. Nos. 5,705,464, 5,710,115, 5,576,282, 4,321,165 and4,284,532. Preferred builders for use in liquid detergents herein aredescribed in U.S. Pat. Nos. 5,705,464, 5,710,115, 5,576,282 and4,284,532.

Inorganic Builders—The detergent compositions herein may also optionallycontain one or more types of inorganic detergent builders beyond thoselisted hereinbefore that also finction as alkalinity sources. Suchoptional inorganic builders can include, for example, aluminosilicatessuch as zeolites. Aluminosilicate zeolites, and their use as detergentbuilders are more fully discussed in Corkill et al., U.S. Pat. No.4,605,509; Issued Aug. 12, 1986, the disclosure of which is incorporatedherein by reference. Also crystalline layered silicates, such as thosediscussed in this '509 U.S. patent, are also suitable for use in thedetergent compositions herein. If utilized, optional inorganic detergentbuilders can comprise from about 2% to 15% by weight of the compositionsherein. Additional examples of inorganic builders are described in U.S.Pat. Nos. 5,705,464 and 5,710,115.

Surfactants—Detersive surfactants included in the fully-formulateddetergent compositions afforded by the present invention comprises atleast 0.01%, preferably from about 0.5% to about 50%, by weight ofdetergent composition depending upon the particular surfactants used andthe desired effects. In a highly preferred embodiment, the detersivesurfactant comprises from about 0.5% to about 20% by weight of thecomposition.

The detersive surfactant can be nonionic, anionic, ampholytic,zwitterionic, or cationic nonlimiting examples of which are disclosed inU.S. Pat. Nos. 5,707,950 and 5,576,282. Mixtures of these surfactantscan also be used. Preferred detergent compositions comprise anionicdetersive surfactants or mixtures of anionic surfactants with othersurfactants, especially nonionic surfactants.

Nonlimiting examples of surfactants useful herein include theconventional C₁₁-C₁₈ alkyl benzene sulfonates and primary, secondary andrandom alkyl sulfates, the C₁₀-C₁₈ alkyl alkoxy sulfates, the C₁₀-C₁₈alkyl polyglycosides and their corresponding sulfated polyglycosides,C₁₂-C₁₈ alpha-sulfonated fatty acid esters, C₁₂-C₁₈ alkyl and alkylphenol alkoxylates (especially ethoxylates and mixed ethoxy/propoxy),C₁₂-C₁₈ betaines and sulfobetaines (“sultaines”), C₁₀-C₁₈ amine oxides,and the like. Other conventional useful surfactants are listed instandard texts.

Particularly preferred surfactants in the preferred automaticdishwashing compositions (ADD) of the present invention are low foamingnonionic surfactants (LFNI) which are described in U.S. Pat. Nos.5,705,464 and 5,710,115. LFNI may be present in amounts from 0.01% toabout 10% by weight, preferably from about 0.1% to about 10%, and mostpreferably from about 0.25% to about 4%. LFNIs are most typically usedin ADDs on account of the improved water-sheeting action (especiallyfrom glass) which they confer to the ADD product. They also encompassnon-silicone, nonphosphate polymeric materials further illustratedhereinafter which are known to defoam food soils encountered inautomatic dishwashing.

Preferred LFNIs include nonionic alkoxylated surfactants, especiallyethoxylates derived from primary alcohols, and blends thereof with moresophisticated surfactants, such as thepolyoxypropylene/polyoxyethylene/polyoxypropylene (PO/EO/PO) reverseblock polymers as described in U.S. Pat. Nos. 5,705,464 and 5,710,115.

Highly preferred ADDs herein wherein the LFNI is present make use ofethoxylated monohydroxy alcohol or alkyl phenol and additionallycomprise a polyoxyethylene, polyoxypropylene block polymeric compound asdescribed in U.S. Pat. Nos. 5,705,464 and 5,710,115.

LFNIs which may also be used include those POLY-TERGENT® SLF-18 nonionicsurfactants from Olin Corp., and any biodegradable LFNI having themelting point properties discussed hereinabove.

These and other nonionic surfactants are well known in the art, beingdescribed in more detail in Kirk Othmer's Encyclopedia of ChemicalTechnology, 3rd Ed., Vol. 22, pp. 360-379, “Surfactants and DetersiveSystems”, incorporated by reference herein.

Preferred are ADD compositions comprising mixed surfactants wherein thesudsing (absent any silicone suds controlling agent) is less than 2inches, preferably less than 1 inch, as determined by the disclosurebelow.

The equipment useful for these measurements are: a Whirlpool Dishwasher(model 900) equipped with clear plexiglass door, IBM computer datacollection with Labview and Excel Software, proximity sensor (NewarkCorp.—model 95F5203) using SCXI interface, and a plastic ruler.

The data is collected as follows. The proximity sensor is affixed to thebottom dishwasher rack on a metal bracket. The sensor faces downwardtoward the rotating dishwasher arm on the bottom of the machine(distance approximately 2 cm. from the rotating arm). Each pass of therotating arm is measured by the proximity sensor and recorded. Thepulses recorded by the computer are converted to rotations per minute(RPM) of the bottom arm by counting pulses over a 30 second interval.The rate of the arm rotation is directly proportional to the amount ofsuds in the machine and in the dishwasher pump (i.e., the more sudsproduced, the slower the arm rotation).

The plastic ruler is clipped to the bottom rack of the dishwasher andextends to the floor of the machine. At the end of the wash cycle, theheight of the suds is measured using the plastic ruler (viewed throughthe clear door) and recorded as suds height.

The following procedure is followed for evaluating ADD compositions forsuds production as well as for evaluating nonionic surfactants forutility. (For separate evaluation of nonionic surfactant, a base ADDformula, such as Cascade powder, is used along with the nonionicsurfactants which are added separately in glass vials to the dishwashingmachine.)

First, the machine is filled with water (adjust water for appropriatetemperature and hardness) and proceed through a rinse cycle. The RPM ismonitored throughout the cycle (approximately 2 min.) without any ADDproduct (or surfactants) being added (a quality control check to ensurethe machine is functioning properly). As the machine begins to fill forthe wash cycle, the water is again adjusted for temperature andhardness, and then the ADD product is added to the bottom of the machine(in the case of separately evaluated surfactants, the ADD base formulais first added to the bottom of the machine then the surfactants areadded by placing the surfactant-containing glass vials inverted on thetop rack of the machine). The RPM is then monitored throughout the washcycle. At the end of the wash cycle, the suds height is recorded usingthe plastic ruler. The machine is again filled with water (adjust waterfor appropriate temperature and hardness) and runs through another rinsecycle. The RPM is monitored throughout this cycle.

An average RPM is calculated for the 1st rinse, main wash, and finalrinse. The % RPM efficiency is then calculated by dividing the averageRPM for the test surfactants into the average RPM for the control system(base ADD formulation without the nonionic surfactant). The RPMefficiency and suds height measurements are used to dimension theoverall suds profile of the surfactant.

Bleaching Agents—Hydrogen peroxide sources are described in detail inthe herein incorporated Kirk Othmer's Encyclopedia of ChemicalTechnology, 4th Ed (1992, John Wiley & Sons), Vol. 4, pp. 271-300“Bleaching Agents (Survey)”, and include the various forms of sodiumperborate and sodium percarbonate, including various coated and modifiedforms. An “effective amount” of a source of hydrogen peroxide is anyamount capable of measurably improving stain removal (especially of teastains) from soiled dishware compared to a hydrogen peroxide source-freecomposition when the soiled dishware is washed by the consumer in adomestic automatic dishwasher in the presence of alkali.

More generally a source of hydrogen peroxide herein is any convenientcompound or mixture which under consumer use conditions provides aneffective amount of hydrogen peroxide. Levels may vary widely and areusually in the range from about 0.1% to about 70%, more typically fromabout 0.5% to about 30%, by weight of the ADD compositions herein.

The preferred source of hydrogen peroxide used herein can be anyconvenient source, including hydrogen peroxide itself. For example,perborate, e.g., sodium perborate (any hydrate but preferably the mono-or tetra-hydrate), sodium carbonate peroxyhydrate or equivalentpercarbonate salts, sodium pyrophosphate peroxyhydrate, ureaperoxyhydrate, or sodium peroxide can be used herein. Also useful aresources of available oxygen such as persulfate bleach (e.g., OXONE,manufactured by DuPont). Sodium perborate monohydrate and sodiumpercarbonate are particularly preferred. Mixtures of any convenienthydrogen peroxide sources can also be used.

A preferred percarbonate bleach comprises dry particles having anaverage particle size in the range from about 500 micrometers to about1,000 micrometers, not more than about 10% by weight of said particlesbeing smaller than about 200 micrometers and not more than about 10% byweight of said particles being larger than about 1,250 micrometers.Optionally, the percarbonate can be coated with a silicate, borate orwater-soluble surfactants. Percarbonate is available from variouscommercial sources such as FMC, Solvay and Tokai Denka.

While not preferred for ADD compositions of the present invention whichcomprise detersive enzymes, the present invention compositions may alsocomprise as the bleaching agent a chlorine-type bleaching material. Suchagents are well known in the art, and include for example sodiumdichloroisocyanurate (“NaDCC”).

While effective ADD compositions herein may comprise only the nonionicsurfactant and builder, fully-formulated ADD compositions typically willalso comprise other automatic dishwashing detergent adjunct materials toimprove or modify performance. These materials are selected asappropriate for the properties required of an automatic dishwashingcomposition. For example, low spotting and filming is desired—preferredcompositions have spotting and filming grades of 3 or less, preferablyless than 2, and most preferably less than 1, as measured by thestandard test of The American Society for Testing and Materials (“ASTM”)D3556-85 (Reapproved 1989) “Standard Test Method for Deposition onGlassware During Mechanical Dishwashing”.

(a) Bleach Activators—Preferably, the peroxygen bleach component in thecomposition is formulated with an activator (peracid precursor). Theactivator is present at levels of from about 0.01% to about 15%,preferably from about 0.5% to about 10%, more preferably from about 1%to about 8%, by weight of the composition. Preferred activators areselected from the group consisting of tetraacetyl ethylene diamine(TAED), benzoylcaprolactam (BzCL), 4-nitrobenzoylcaprolactam,3-chlorobenzoylcaprolactam, benzoyloxybenzenesulphonate (BOBS),nonanoyloxybenzenesulphonate (NOBS), phenyl benzoate (PhBz),decanoyloxybenzenesulphonate (C₁₀-OBS), benzoylvalerolactam (BZVL),octanoyloxybenzenesulphonate (C₈-OBS), perhydrolyzable esters andmixtures thereof, most preferably benzoylcaprolactam andbenzoylvalerolactam. Particularly preferred bleach activators in the pHrange from about 8 to about 9.5 are those selected having an OBS or VLleaving group.

Preferred bleach activators are those described in U.S. Pat. Nos.5,698,504, 5,695,679, 5,686,014, 5,130,045 and 4,412,934, and copendingpatent applications U.S. Ser. Nos. 08/064,624, 08/064,623, 08/064,621,08/064,562, 08/064,564, 08/082,270 and copending application to M.Burns, A. D. Willey, R. T. Hartshorn, C. K. Ghosh, entitled “BleachingCompounds Comprising Peroxyacid Activators Used With Enzymes” and havingU.S. Ser. No. 08/133,691 (P&G Case 4890R), all of which are incorporatedherein by reference.

The mole ratio of peroxygen bleaching compound (as AvO) to bleachactivator in the present invention generally ranges from at least 1:1,preferably from about 20:1 to about 1:1, more preferably from about 10:1to about 3:1.

Quaternary substituted bleach activators may also be included. Thepresent detergent compositions preferably comprise a quaternarysubstituted bleach activator (QSBA) or a quaternary substituted peracid(QSP); more preferably, the former. Preferred QSBA structures arefurther described in copending U.S. Ser. No. 08/298,903, 08/298,650,08/298,906 and 08/298,904 filed Aug. 31, 1994, now respectively U.S.Pat. Nos. 5,686,015, 5,460,747, 5,584,888 and 5,578,136, incorporatedherein by reference.

Highly preferred bleach activators useful herein are amide-substitutedas described in U.S. Pat. Nos. 5,698,504, 5,695,679 and 5,686,014.Preferred examples of such bleach activators include:(6-octanamidocaproyl) oxybenzenesulfonate,(6-nonanamidocaproyl)oxybenzenesulfonate,(6-decanamidocaproyl)oxybenzenesulfonate and mixtures thereof.

Other useful activators, disclosed in U.S. Pat. Nos. 5,698,504,5,695,679, 5,686,014 and 4,966,723, include benzoxazin-type activators,such as a C₆H₄ ring to which is fused in the 1,2-positions a moiety—C(O)OC(R¹)═N—.

Depending on the activator and precise application, good bleachingresults can be obtained from bleaching systems having with in-use pH offrom about 6 to about 13, preferably from about 9.0 to about 10.5.Typically, for example, activators with electron-withdrawing moietiesare used for near-neutral or sub-neutral pH ranges. Alkalis andbuffering agents can be used to secure such pH.

Acyl lactam activators, as described in U.S. Pat. Nos. 5,698,504,5,695,679 and 5,686,014, are very useful herein, especially the acylcaprolactams (see for example WO 94-28102 A) and acyl valerolactams (seeU.S. Pat. No. 5,503,639).

(b) Organic Peroxides, especially Diacyl Peroxides—These are extensivelyillustrated in Kirk Othmer, Encyclopedia of Chemical Technology, Vol.17, John Wiley and Sons, 1982 at pages 27-90 and especially at pages63-72, all incorporated herein by reference. If a diacyl peroxide isused, it will preferably be one which exerts minimal adverse impact onspotting/filming.

(c) Metal-containing Bleach Catalysts—The present invention compositionsand methods utilize metal-containing bleach catalysts that are effectivefor use in ADD compositions. Preferred are manganese andcobalt-containing bleach catalysts.

One type of metal-containing bleach catalyst is a catalyst systemcomprising a transition metal cation of defined bleach catalyticactivity, such as copper, iron, titanium, ruthenium tungsten,molybdenum, or manganese cations, an auxiliary metal cation havinglittle or no bleach catalytic activity, such as zinc or aluminumcations, and a sequestrate having defined stability constants for thecatalytic and auxiliary metal cations, particularlyethylenediaminetetraacetic acid, ethylenediaminetetra(methylenephosphonic acid) and water-soluble salts thereof. Suchcatalysts are disclosed in U.S. Pat. No. 4,430,243.

If desired, the compositions herein can be catalyzed by means of amanganese compound. Such compounds and levels of use are well known inthe art and include, for example, the manganese-based catalystsdisclosed in U.S. Pat. Nos. 5,576,282, 5,246,621, 5,244,594; 5,194,416;5,114,606; and European Pat. App. Pub. Nos. 549,271A1, 549,272A1,544,440A2, and 544,490A1; Preferred examples of these catalysts includeMn^(IV) ₂(u-O)₃(1,4,7-trimethyl-1,4,7-triazacyclononane)₂(PF₆)₂,Mn^(III) ₂(u-O)₁(u-OAc)₂(1,4,7-trimethyl-1,4,7-triazacyclononane)₂(CIO₄)₂, Mn^(IV)₄(u-O)₆(1,4,7-triazacyclononane)₄(CIO₄)₄, Mn^(III)Mn^(IV)₄(u-O)₁(u-OAc)₂-(1,4,7-trimethyl-1,4,7-triazacyclononane)₂(CIO₄)₃,Mn^(IV)(1,4,7-trimethyl-1,4,7-triazacyclononane)-(OCH₃)₃(PF₆), andmixtures thereof. Other metal-based bleach catalysts include thosedisclosed in U.S. Pat. Nos. 4,430,243 and 5,114,611. The use ofmanganese with various complex ligands to enhance bleaching is alsoreported in the following U.S. Pat. Nos: 4,728,455; 5,284,944;5,246,612; 5,256,779; 5,280,117; 5,274,147; 5,153,161; and 5,227,084.

Cobalt bleach catalysts useful herein are known, and are described, forexample, in U.S. Pat. Nos. 5,597,936, 5,595,967, 5,703,030 and M. L.Tobe, “Base Hydrolysis of Transition-Metal Complexes”, Adv. Inorg.Bioinorg. Mech., (1983), 2, pages 1-94. The most preferred cobaltcatalyst useful herein are cobalt pentaamine acetate salts having theformula [Co(NH₃)₅OAc] T_(y), wherein “OAc” represents an acetate moietyand “T_(y)” is an anion, and especially cobalt pentaamine acetatechloride, [Co(NH₃)₅OAc]Cl₂; as well as [Co(NH₃)₅OAc](OAc)₂;[Co(NH₃)₅OAc](PF₆)₂; [Co(NH₃)₅OAc](SO₄); [Co(NH₃)₅OAc](BF₄)₂; and[Co(NH₃)₅OAc](NO₃)₂ (herein “PAC”).

These cobalt catalysts are readily prepared by known procedures, such astaught for example in U.S. Pat. Nos. 5,597,936, 5,595,967, 5,703,030, inthe Tobe article and the references cited therein, and in U.S. Pat. No.4,810,410, to Diakun et al, issued Mar. 7,1989, J. Chem. Ed. (1989), 66(12), 1043-45; The Synthesis and Characterization of InorganicCompounds, W. L. Jolly (Prentice-Hall; 1970), pp. 461-3; Inorg. Chem.,18, 1497-1502 (1979); Inorg. Chem., 21, 2881-2885 (1982); Inorg. Chem.,18, 2023-2025 (1979); Inorg. Synthesis, 173-176 (1960); and Journal ofPhysical Chemistry 56, 22-25 (1952).

The bleach catalysts useful in automatic dishwashing compositions andconcentrated powder detergent compositions may also be selected asappropriate for the present invention. For examples of suitable bleachcatalysts see U.S. Pat. Nos. 4,246,612 and 5,227,084.

As a practical matter, and not by way of limitation, the compositionsand cleaning processes herein can be adjusted to provide on the order ofat least one part per hundred million of the active bleach catalystspecies in the aqueous washing medium, and will preferably provide fromabout 0.01 ppm to about 25 ppm, more preferably from about 0.05 ppm toabout 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, ofthe bleach catalyst species in the wash liquor. In order to obtain suchlevels in the wash liquor of an automatic washing process, typicalcompositions herein will comprise from about 0.0005% to about 0.2%, morepreferably from about 0.004% to about 0.08%, of bleach catalyst,especially manganese or cobalt catalysts, by weight of the cleaningcompositions.

pH and Buffering Variation—Many detergent compositions herein will bebuffered, i.e., they are relatively resistant to pH drop in the presenceof acidic soils. However, other compositions herein may haveexceptionally low buffering capacity, or may be substantiallyunbuffered. Techniques for controlling or varying pH at recommendedusage levels more generally include the use of not only buffers, butalso additional alkalis, acids, pH-jump systems, dual compartmentcontainers, etc., and are well known to those skilled in the art.

The preferred ADD compositions herein comprise a pH-adjusting componentselected from water-soluble alkaline inorganic salts and water-solubleorganic or inorganic builders as described in U.S. Pat. Nos. 5,705,464and 5,710,115.

Water-Soluble Silicates—The present automatic dishwashing detergentcompositions may further comprise water-soluble silicates as describedin U.S. Pat. Nos. 5,705,464 and 5,710,115.

Material Care Agents—The preferred ADD compositions may contain one ormore material care agents which are effective as corrosion inhibitorsand/or anti-tarnish aids as described in U.S. Pat. Nos. 5,705,464,5,710,115 and 5,646,101. When present, such protecting materials arepreferably incorporated at low levels, e.g., from about 0.01% to about5% of the ADD composition.

Other Materials—Detersive ingredients or adjuncts optionally included inthe instant compositions can include one or more materials for assistingor enhancing cleaning performance, treatment of the substrate to becleaned, or designed to improve the aesthetics of the compositions.Adjuncts which can also be included in compositions of the presentinvention, at their conventional art-established levels for use(generally, adjunct materials comprise, in total, from about 30% toabout 99.9%, preferably from about 70% to about 95%, by weight of thecompositions), include other active ingredients such as non-phosphatebuilders, chelants, enzymes, suds suppressors, dispersant polymers(e.g., from BASF Corp. or Rohm & Haas), color speckles, silvercare,anti-tarnish and/or anti-corrosion agents, dyes, fillers, germicides,alkalinity sources, hydrotropes, anti-oxidants, enzyme stabilizingagents, perfiunes, solubilizing agents, carriers, processing aids,pigments, and pH control agents as described in U.S. Pat. Nos.5,705,464, 5,710,115, 5,698,504, 5,695,679, 5,686,014 and 5,646,101.

The following nonlimiting examples further illustrate the ADDcompositions of the present invention.

EXAMPLE 1

Weight Ingredients A B Sodium Tripolyphosphate (STPP) 24.0 45 Sodiumcarbonate 20.0 13.5 Hydrated 2.0 r silicate 15 13.5 nonionic surfactants2.0 2.0 Polymer¹ 4.0 — Protease² (4% active) 0.83 0.83 Amylase (0.8%active) 0.5 0.5 Perborate monohydrate (15.5% Active AvO)³ 14.5 14.5Cobalt catalyst⁴ 0.008 — Dibenzoyl Peroxide (18% active) 4.4 4.4 Water,sodium sulfate and misc. Balance Balance ¹Terpolymer selected fromeither 60% acrylic acid/20% maleic acid/20% ethyl acrylate, or 70%acrylic acid/10% maleic acid/20% ethyl acrylate. ²A carbonyl hydrolasevariant of B. amyloliquefaciens subtilisin with the amino acidsubstitutions 210I/76D/103A/104I/156E/166D. ³Available from DeGussaCorp. The AvO level of the above formula is 2.2%.⁴Pentaammineacetatocobalt(III) nitrate prepared as describedhereinbefore; may be replaced by MnTACN.

The ADD's of the above dishwashing detergent composition examples areused to wash milk-soiled glasses, starch, cheese, egg or babyfood-soiled flatware, by loading the soiled dishes in a domestic automaticdishwashing appliance and washing using either cold fill, 60° C. peak,or uniformly 45-50° C. wash cycles with a product concentration of theexemplary compositions of from about 1,000 to about 8,000 ppm, withexcellent results.

EXAMPLE 2

Light-duty liquid dishwashing detergent formulae are prepared asfollows:

Composition A B C Ingredient % Weight Surfactant 32.00 29.50 30.75Ethanol 4.00 4.00 4.00 Ammonium citrate 0.06 0.06 0.06 Magnesiumchloride 3.32 3.32 3.32 Ammonium sulfate 0.08 0.08 0.08 Hydrogenperoxide 200 ppm 200 ppm 200 ppm Perfume 0.18 0.18 0.18 Protease¹ 0.500.50 0.50 Water and minors Balance ¹A carbonyl hydrolase variant of B.amyloliquefaciens subtilisin with the amino acid substitutions210I/76D/103A/104I/156E/166D.

Having described the invention in detail with reference to preferredembodiments and the examples, it will be clear to those skilled in theart that various changes and modifications may be made without departingfrom the scope of the invention and the invention is not to beconsidered limited to what is described in the specification.

9 1497 base pairs nucleic acid single linear DNA (genomic) 1 GGTCTACTAAAATATTATTC CATACTATAC AATTAATACA CAGAATAATC TGTCTATTGG 60 TTATTCTGCAAATGAAAAAA AGGAGAGGAT AAAGAGTGAG AGGCAAAAAA GTATGGATCA 120 GTTTGCTGTTTGCTTTAGCG TTAATCTTTA CGATGGCGTT CGGCAGCACA TCCTCTGCCC 180 AGGCGGCAGGGAAATCAAAC GGGGAAAAGA AATATATTGT CGGGTTTAAA CAGACAATGA 240 GCACGATGAGCGCCGCTAAG AAGAAAGATG TCATTTCTGA AAAAGGCGGG AAAGTGCAAA 300 AGCAATTCAAATATGTAGAC GCAGCTTCAG TCACATTAAA CGAAAAAGCT GTAAAAGAAT 360 TGAAAAAAGACCCGAGCGTC GCTTACGTTG AAGAAGATCA CGTAGCACAT GCGTACGCGC 420 AGTCCGTGCCTTACGGCGTA TCACAAATTA AAGCCCCTGC TCTGCACTCT CAAGGCTACA 480 CTGGATCAAATGTTAAAGTA GCGGTTATCG ACAGCGGTAT CGATTCTTCT CATCCTGATT 540 TAAAGGTAGCAAGCGGAGCC AGCATGGTTC CTTCTGAAAC AAATCCTTTC CAAGACAACA 600 ACTCTCACGGAACTCACGTT GCCGGCACAG TTGCGGCTCT TAATAACTCA ATCGGTGTAT 660 TAGGCGTTGCGCCAAGCGCA TCACTTTACG CTGTAAAAGT TCTCGGTGCT GACGGTTCCG 720 GCCAATACAGCTGGATCATT AACGGAATCG AGTGGGCGAT CGCAAACAAT ATGGACGTTA 780 TTAACATGAGCCTCGGCGGA CCTTCTGGTT CTGCTGCTTT AAAAGCGGCA GTTGATAAAG 840 CCGTTGCATCCGGCGTCGTA GTCGTTGCGG CAGCCGGTAA CGAAGGCACT TCCGGCAGCT 900 CAAGCACAGTGGGCTACCCT GGTAAATACC CTTCTGTCAT TGCAGTAGGC GCTGTTGACA 960 GCAGCAACCAAAGAGCATCT TTCTCAAGCG TAGGACCTGA GCTTGATGTC ATGGCACCTG 1020 GCGTATCTATCCAAAGCACG CTTCCTGGAA ACAAATACGG GGCGTACAAC GGTACGTCAA 1080 TGGCATCTCCGCACGTTGCC GGAGCGGCTG CTTTGATTCT TTCTAAGCAC CCGAACTGGA 1140 CAAACACTCAAGTCCGCAGC AGTTTAGAAA ACACCACTAC AAAACTTGGT GATTCTTTGT 1200 ACTATGGAAAAGGGCTGATC AACGTACAAG CGGCAGCTCA GTAAAACATA AAAAACCGGC 1260 CTTGGCCCCGCCGGTTTTTT ATTATTTTTC TTCCTCCGCA TGTTCAATCC GCTCCATAAT 1320 CGACGGATGGCTCCCTCTGA AAATTTTAAC GAGAAACGGC GGGTTGACCC GGCTCAGTCC 1380 CGTAACGGCCAACTCCTGAA ACGTCTCAAT CGCCGCTTCC CGGTTTCCGG TCAGCTCAAT 1440 GCCATAACGGTCGGCGGCGT TTTCCTGATA CCGGGAGACG GCATTCGTAA TCGGATC 1497 275 amino acidsamino acid single linear protein 2 Ala Gln Ser Val Pro Tyr Gly Val SerGln Ile Lys Ala Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly SerAsn Val Lys Val Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His ProAsp Leu Lys Val Ala Gly Gly Ala 35 40 45 Ser Met Val Pro Ser Glu Thr AsnPro Phe Gln Asp Asn Asn Ser His 50 55 60 Gly Thr His Val Ala Gly Thr ValAla Ala Leu Asn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ala Pro SerAla Ser Leu Tyr Ala Val Lys Val Leu 85 90 95 Gly Ala Asp Gly Ser Gly GlnTyr Ser Trp Ile Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ala Asn AsnMet Asp Val Ile Asn Met Ser Leu Gly Gly 115 120 125 Pro Ser Gly Ser AlaAla Leu Lys Ala Ala Val Asp Lys Ala Val Ala 130 135 140 Ser Gly Val ValVal Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly 145 150 155 160 Ser SerSer Thr Val Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala 165 170 175 ValGly Ala Val Asp Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val 180 185 190Gly Pro Glu Leu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200205 Leu Pro Gly Asn Lys Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser 210215 220 Pro His Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn225 230 235 240 Trp Thr Asn Thr Gln Val Arg Ser Ser Leu Glu Asn Thr ThrThr Lys 245 250 255 Leu Gly Asp Ser Phe Tyr Tyr Gly Lys Gly Leu Ile AsnVal Gln Ala 260 265 270 Ala Ala Gln 275 275 amino acids amino acidsingle linear protein 3 Ala Gln Ser Val Pro Tyr Gly Ile Ser Gln Ile LysAla Pro Ala Leu 1 5 10 15 His Ser Gln Gly Tyr Thr Gly Ser Asn Val LysVal Ala Val Ile Asp 20 25 30 Ser Gly Ile Asp Ser Ser His Pro Asp Leu AsnVal Arg Gly Gly Ala 35 40 45 Ser Phe Val Pro Ser Glu Thr Asn Pro Tyr GlnAsp Gly Ser Ser His 50 55 60 Gly Thr His Val Ala Gly Thr Ile Ala Ala LeuAsn Asn Ser Ile Gly 65 70 75 80 Val Leu Gly Val Ser Pro Ser Ala Ser LeuTyr Ala Val Lys Val Leu 85 90 95 Asp Ser Thr Gly Ser Gly Gln Tyr Ser TrpIle Ile Asn Gly Ile Glu 100 105 110 Trp Ala Ile Ser Asn Asn Met Asp ValIle Asn Met Ser Leu Gly Gly 115 120 125 Pro Thr Gly Ser Thr Ala Leu LysThr Val Val Asp Lys Ala Val Ser 130 135 140 Ser Gly Ile Val Val Ala AlaAla Ala Gly Asn Glu Gly Ser Ser Gly 145 150 155 160 Ser Thr Ser Thr ValGly Tyr Pro Ala Lys Tyr Pro Ser Thr Ile Ala 165 170 175 Val Gly Ala ValAsn Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Ala 180 185 190 Gly Ser GluLeu Asp Val Met Ala Pro Gly Val Ser Ile Gln Ser Thr 195 200 205 Leu ProGly Gly Thr Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Thr 210 215 220 ProHis Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Thr 225 230 235240 Trp Thr Asn Ala Gln Val Arg Asp Arg Leu Glu Ser Thr Ala Thr Tyr 245250 255 Leu Gly Asn Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala260 265 270 Ala Ala Gln 275 274 amino acids amino acid single linearprotein 4 Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp LysVal 1 5 10 15 Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala ValLeu Asp 20 25 30 Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val GlyGly Ala 35 40 45 Ser Phe Val Ala Gly Glu Ala Tyr Asn Thr Asp Gly Asn GlyHis Gly 50 55 60 Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr ThrGly Val 65 70 75 80 Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val LysVal Leu Asn 85 90 95 Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser GlyIle Glu Trp 100 105 110 Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met SerLeu Gly Gly Ala 115 120 125 Ser Gly Ser Thr Ala Met Lys Gln Ala Val AspAsn Ala Tyr Ala Arg 130 135 140 Gly Val Val Val Val Ala Ala Ala Gly AsnSer Gly Asn Ser Gly Ser 145 150 155 160 Thr Asn Thr Ile Gly Tyr Pro AlaLys Tyr Asp Ser Val Ile Ala Val 165 170 175 Gly Ala Val Asp Ser Asn SerAsn Arg Ala Ser Phe Ser Ser Val Gly 180 185 190 Ala Glu Leu Glu Val MetAla Pro Gly Ala Gly Val Tyr Ser Thr Tyr 195 200 205 Pro Thr Asn Thr TyrAla Thr Leu Asn Gly Thr Ser Met Ala Ser Pro 210 215 220 His Val Ala GlyAla Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu 225 230 235 240 Ser AlaSer Gln Val Arg Asn Arg Leu Ser Ser Thr Ala Thr Tyr Leu 245 250 255 GlySer Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala 260 265 270Ala Gln 269 amino acids amino acid single linear protein 5 Ala Gln SerVal Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala 1 5 10 15 His AsnArg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp 20 25 30 Thr GlyIle Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser 35 40 45 Phe ValPro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr 50 55 60 His ValAla Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu 65 70 75 80 GlyVal Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala 85 90 95 SerGly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala 100 105 110Gly Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser 115 120125 Pro Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly 130135 140 Val Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala Gly Ser Ile Ser145 150 155 160 Tyr Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala ThrAsp Gln 165 170 175 Asn Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala GlyLeu Asp Ile 180 185 190 Val Ala Pro Gly Val Asn Val Gln Ser Thr Tyr ProGly Ser Thr Tyr 195 200 205 Ala Ser Leu Asn Gly Thr Ser Met Ala Thr ProHis Val Ala Gly Ala 210 215 220 Ala Ala Leu Val Lys Gln Lys Asn Pro SerTrp Ser Asn Val Gln Ile 225 230 235 240 Arg Asn His Leu Lys Asn Thr AlaThr Ser Leu Gly Ser Thr Asn Leu 245 250 255 Tyr Gly Ser Gly Leu Val AsnAla Glu Ala Ala Thr Arg 260 265 1140 base pairs nucleic acid singlelinear DNA (genomic) CDS 1..1140 6 ATG AAG AAA CCG TTG GGG AAA ATT GTCGCA AGC ACC GCA CTA CTC ATT 48 Met Lys Lys Pro Leu Gly Lys Ile Val AlaSer Thr Ala Leu Leu Ile 1 5 10 15 TCT GTT GCT TTT AGT TCA TCG ATC GCATCG GCT GCT GAA GAA GCA AAA 96 Ser Val Ala Phe Ser Ser Ser Ile Ala SerAla Ala Glu Glu Ala Lys 20 25 30 GAA AAA TAT TTA ATT GGC TTT AAT GAG CAGGAA GCT GTC AGT GAG TTT 144 Glu Lys Tyr Leu Ile Gly Phe Asn Glu Gln GluAla Val Ser Glu Phe 35 40 45 GTA GAA CAA GTA GAG GCA AAT GAC GAG GTC GCCATT CTC TCT GAG GAA 192 Val Glu Gln Val Glu Ala Asn Asp Glu Val Ala IleLeu Ser Glu Glu 50 55 60 GAG GAA GTC GAA ATT GAA TTG CTT CAT GAA TTT GAAACG ATT CCT GTT 240 Glu Glu Val Glu Ile Glu Leu Leu His Glu Phe Glu ThrIle Pro Val 65 70 75 80 TTA TCC GTT GAG TTA AGC CCA GAA GAT GTG GAC GCGCTT GAA CTC GAT 288 Leu Ser Val Glu Leu Ser Pro Glu Asp Val Asp Ala LeuGlu Leu Asp 85 90 95 CCA GCG ATT TCT TAT ATT GAA GAG GAT GCA GAA GTA ACGACA ATG GCG 336 Pro Ala Ile Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr ThrMet Ala 100 105 110 CAA TCA GTG CCA TGG GGA ATT AGC CGT GTG CAA GCC CCAGCT GCC CAT 384 Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro AlaAla His 115 120 125 AAC CGT GGA TTG ACA GGT TCT GGT GTA AAA GTT GCT GTCCTC GAT ACA 432 Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val LeuAsp Thr 130 135 140 GGT ATT TCC ACT CAT CCA GAC TTA AAT ATT CGT GGT GGCGCT AGC TTT 480 Gly Ile Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly AlaSer Phe 145 150 155 160 GTA CCA GGG GAA CCA TCC ACT CAA GAT GGG AAT GGGCAT GGC ACG CAT 528 Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly HisGly Thr His 165 170 175 GTG GCC GGG ACG ATT GCT GCT TTA AAC AAT TCG ATTGGC GTT CTT GGC 576 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile GlyVal Leu Gly 180 185 190 GTA GCG CCG AGC GCG GAA CTA TAC GCT GTT AAA GTATTA GGG GCG AGC 624 Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val LeuGly Ala Ser 195 200 205 GGT TCA GGT TCG GTC AGC TCG ATT GCC CAA GGA TTGGAA TGG GCA GGG 672 Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu GluTrp Ala Gly 210 215 220 AAC AAT GGC ATG CAC GTT GCT AAT TTG AGT TTA GGAAGC CCT TCG CCA 720 Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly SerPro Ser Pro 225 230 235 240 AGT GCC ACA CTT GAG CAA GCT GTT AAT AGC GCGACT TCT AGA GGC GTT 768 Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala ThrSer Arg Gly Val 245 250 255 CTT GTT GTA GCG GCA TCT GGG AAT TCA GGT GCAGGC TCA ATC AGC TAT 816 Leu Val Val Ala Ala Ser Gly Asn Ser Gly Ala GlySer Ile Ser Tyr 260 265 270 CCG GCC CGT TAT GCG AAC GCA ATG GCA GTC GGAGCT ACT GAC CAA AAC 864 Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly AlaThr Asp Gln Asn 275 280 285 AAC AAC CGC GCC AGC TTT TCA CAG TAT GGC GCAGGG CTT GAC ATT GTC 912 Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala GlyLeu Asp Ile Val 290 295 300 GCA CCA GGT GTA AAC GTG CAG AGC ACA TAC CCAGGT TCA ACG TAT GCC 960 Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro GlySer Thr Tyr Ala 305 310 315 320 AGC TTA AAC GGT ACA TCG ATG GCT ACT CCTCAT GTT GCA GGT GCA GCA 1008 Ser Leu Asn Gly Thr Ser Met Ala Thr Pro HisVal Ala Gly Ala Ala 325 330 335 GCC CTT GTT AAA CAA AAG AAC CCA TCT TGGTCC AAT GTA CAA ATC CGC 1056 Ala Leu Val Lys Gln Lys Asn Pro Ser Trp SerAsn Val Gln Ile Arg 340 345 350 AAT CAT CTA AAG AAT ACG GCA ACG AGC TTAGGA AGC ACG AAC TTG TAT 1104 Asn His Leu Lys Asn Thr Ala Thr Ser Leu GlySer Thr Asn Leu Tyr 355 360 365 GGA AGC GGA CTT GTC AAT GCA GAA GCG GCAACA CGC 1140 Gly Ser Gly Leu Val Asn Ala Glu Ala Ala Thr Arg 370 375 380380 amino acids amino acid linear protein 7 Met Lys Lys Pro Leu Gly LysIle Val Ala Ser Thr Ala Leu Leu Ile 1 5 10 15 Ser Val Ala Phe Ser SerSer Ile Ala Ser Ala Ala Glu Glu Ala Lys 20 25 30 Glu Lys Tyr Leu Ile GlyPhe Asn Glu Gln Glu Ala Val Ser Glu Phe 35 40 45 Val Glu Gln Val Glu AlaAsn Asp Glu Val Ala Ile Leu Ser Glu Glu 50 55 60 Glu Glu Val Glu Ile GluLeu Leu His Glu Phe Glu Thr Ile Pro Val 65 70 75 80 Leu Ser Val Glu LeuSer Pro Glu Asp Val Asp Ala Leu Glu Leu Asp 85 90 95 Pro Ala Ile Ser TyrIle Glu Glu Asp Ala Glu Val Thr Thr Met Ala 100 105 110 Gln Ser Val ProTrp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His 115 120 125 Asn Arg GlyLeu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr 130 135 140 Gly IleSer Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe 145 150 155 160Val Pro Gly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His 165 170175 Val Ala Gly Thr Ile Ala Ala Leu Asn Asn Ser Ile Gly Val Leu Gly 180185 190 Val Ala Pro Ser Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser195 200 205 Gly Ser Gly Ser Val Ser Ser Ile Ala Gln Gly Leu Glu Trp AlaGly 210 215 220 Asn Asn Gly Met His Val Ala Asn Leu Ser Leu Gly Ser ProSer Pro 225 230 235 240 Ser Ala Thr Leu Glu Gln Ala Val Asn Ser Ala ThrSer Arg Gly Val 245 250 255 Leu Val Val Ala Ala Ser Gly Asn Ser Gly AlaGly Ser Ile Ser Tyr 260 265 270 Pro Ala Arg Tyr Ala Asn Ala Met Ala ValGly Ala Thr Asp Gln Asn 275 280 285 Asn Asn Arg Ala Ser Phe Ser Gln TyrGly Ala Gly Leu Asp Ile Val 290 295 300 Ala Pro Gly Val Asn Val Gln SerThr Tyr Pro Gly Ser Thr Tyr Ala 305 310 315 320 Ser Leu Asn Gly Thr SerMet Ala Thr Pro His Val Ala Gly Ala Ala 325 330 335 Ala Leu Val Lys GlnLys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 340 345 350 Asn His Leu LysAsn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr 355 360 365 Gly Ser GlyLeu Val Asn Ala Glu Ala Ala Thr Arg 370 375 380 1140 base pairs nucleicacid single linear DNA (genomic) CDS 1..1140 mat_peptide 334..1140 8 ATGAAG AAA CCG TTG GGG AAA ATT GTC GCA AGC ACC GCA CTA CTC ATT 48 Met LysLys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu Leu Ile -111-110 -105-100 TCT GTT GCT TTT AGT TCA TCG ATC GCA TCG GCT GCT GAA GAA GCA AAA 96Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala Ala Glu Glu Ala Lys -95 -90-85 -80 GAA AAA TAT TTA ATT GGC TTT AAT GAG CAG GAA GCT GTC AGT GAG TTT144 Glu Lys Tyr Leu Ile Gly Phe Asn Glu Gln Glu Ala Val Ser Glu Phe -75-70 -65 GTA GAA CAA GTA GAG GCA AAT GAC GAG GTC GCC ATT CTC TCT GAG GAA192 Val Glu Gln Val Glu Ala Asn Asp Glu Val Ala Ile Leu Ser Glu Glu -60-55 -50 GAG GAA GTC GAA ATT GAA TTG CTT CAT GAA TTT GAA ACG ATT CCT GTT240 Glu Glu Val Glu Ile Glu Leu Leu His Glu Phe Glu Thr Ile Pro Val -45-40 -35 TTA TCC GTT GAG TTA AGC CCA GAA GAT GTG GAC GCG CTT GAA CTC GAT288 Leu Ser Val Glu Leu Ser Pro Glu Asp Val Asp Ala Leu Glu Leu Asp -30-25 -20 CCA GCG ATT TCT TAT ATT GAA GAG GAT GCA GAA GTA ACG ACA ATG GCG336 Pro Ala Ile Ser Tyr Ile Glu Glu Asp Ala Glu Val Thr Thr Met Ala -15-10 -5 1 CAA TCA GTG CCA TGG GGA ATT AGC CGT GTG CAA GCC CCA GCT GCC CAT384 Gln Ser Val Pro Trp Gly Ile Ser Arg Val Gln Ala Pro Ala Ala His 5 1015 AAC CGT GGA TTG ACA GGT TCT GGT GTA AAA GTT GCT GTC CTC GAT ACA 432Asn Arg Gly Leu Thr Gly Ser Gly Val Lys Val Ala Val Leu Asp Thr 20 25 30GGT ATT TCC ACT CAT CCA GAC TTA AAT ATT CGT GGT GGC GCT AGC TTT 480 GlyIle Ser Thr His Pro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe 35 40 45 GTACCA GGG GAA CCA TCC ACT CAA GAT GGG AAT GGG CAT GGC ACG CAT 528 Val ProGly Glu Pro Ser Thr Gln Asp Gly Asn Gly His Gly Thr His 50 55 60 65 GTGGCC GGG ACG ATT GCT GCT TTA GAC AAC TCG ATT GGC GTT CTT GGC 576 Val AlaGly Thr Ile Ala Ala Leu Asp Asn Ser Ile Gly Val Leu Gly 70 75 80 GTA GCGCCG AGC GCG GAA CTA TAC GCT GTT AAA GTA TTA GGG GCG AGC 624 Val Ala ProSer Ala Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser 85 90 95 GGT TCA GGCGCC ATC AGC TCG ATT GCC CAA GGA TTG GAA TGG GCA GGG 672 Gly Ser Gly AlaIle Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly 100 105 110 AAC AAT GGCATG CAC GTT GCT AAT TTG AGT TTA GGA AGC CCT TCG CCA 720 Asn Asn Gly MetHis Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro 115 120 125 AGT GCC ACACTT GAG CAA GCT GTT AAT AGC GCG ACT TCT AGA GGC GTT 768 Ser Ala Thr LeuGlu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val 130 135 140 145 CTT GTTGTA GCG GCA TCT GGG AAT GAA GGT GCA GGC TCA ATC GAC TAT 816 Leu Val ValAla Ala Ser Gly Asn Glu Gly Ala Gly Ser Ile Asp Tyr 150 155 160 CCG GCCCGT TAT GCG AAC GCA ATG GCA GTC GGA GCT ACT GAC CAA AAC 864 Pro Ala ArgTyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn 165 170 175 AAC AACCGC GCC AGC TTT TCA CAG TAT GGC GCA GGG CTT GAC ATT GTC 912 Asn Asn ArgAla Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val 180 185 190 GCA CCAGGT GTA AAC GTG CAG AGC ACA TAC CCA ATT TCA ACG TAT GCC 960 Ala Pro GlyVal Asn Val Gln Ser Thr Tyr Pro Ile Ser Thr Tyr Ala 195 200 205 AGC TTAAAC GGT ACA TCG ATG GCT ACT CCT CAT GTT GCA GGT GCA GCA 1008 Ser Leu AsnGly Thr Ser Met Ala Thr Pro His Val Ala Gly Ala Ala 210 215 220 225 GCCCTT GTT AAA CAA AAG AAC CCA TCT TGG TCC AAT GTA CAA ATC CGC 1056 Ala LeuVal Lys Gln Lys Asn Pro Ser Trp Ser Asn Val Gln Ile Arg 230 235 240 AATCAT CTA AAG AAT ACG GCA ACG AGC TTA GGA AGC ACG AAC TTG TAT 1104 Asn HisLeu Lys Asn Thr Ala Thr Ser Leu Gly Ser Thr Asn Leu Tyr 245 250 255 GGAAGC GGA CTT GTC AAT GCA GAA GCG GCA ACA CGC 1140 Gly Ser Gly Leu Val AsnAla Glu Ala Ala Thr Arg 260 265 380 amino acids amino acid linearprotein 9 Met Lys Lys Pro Leu Gly Lys Ile Val Ala Ser Thr Ala Leu LeuIle -111 -110 -105 -100 Ser Val Ala Phe Ser Ser Ser Ile Ala Ser Ala AlaGlu Glu Ala Lys -95 -90 -85 -80 Glu Lys Tyr Leu Ile Gly Phe Asn Glu GlnGlu Ala Val Ser Glu Phe -75 -70 -65 Val Glu Gln Val Glu Ala Asn Asp GluVal Ala Ile Leu Ser Glu Glu -60 -55 -50 Glu Glu Val Glu Ile Glu Leu LeuHis Glu Phe Glu Thr Ile Pro Val -45 -40 -35 Leu Ser Val Glu Leu Ser ProGlu Asp Val Asp Ala Leu Glu Leu Asp -30 -25 -20 Pro Ala Ile Ser Tyr IleGlu Glu Asp Ala Glu Val Thr Thr Met Ala -15 -10 -5 1 Gln Ser Val Pro TrpGly Ile Ser Arg Val Gln Ala Pro Ala Ala His 5 10 15 Asn Arg Gly Leu ThrGly Ser Gly Val Lys Val Ala Val Leu Asp Thr 20 25 30 Gly Ile Ser Thr HisPro Asp Leu Asn Ile Arg Gly Gly Ala Ser Phe 35 40 45 Val Pro Gly Glu ProSer Thr Gln Asp Gly Asn Gly His Gly Thr His 50 55 60 65 Val Ala Gly ThrIle Ala Ala Leu Asp Asn Ser Ile Gly Val Leu Gly 70 75 80 Val Ala Pro SerAla Glu Leu Tyr Ala Val Lys Val Leu Gly Ala Ser 85 90 95 Gly Ser Gly AlaIle Ser Ser Ile Ala Gln Gly Leu Glu Trp Ala Gly 100 105 110 Asn Asn GlyMet His Val Ala Asn Leu Ser Leu Gly Ser Pro Ser Pro 115 120 125 Ser AlaThr Leu Glu Gln Ala Val Asn Ser Ala Thr Ser Arg Gly Val 130 135 140 145Leu Val Val Ala Ala Ser Gly Asn Glu Gly Ala Gly Ser Ile Asp Tyr 150 155160 Pro Ala Arg Tyr Ala Asn Ala Met Ala Val Gly Ala Thr Asp Gln Asn 165170 175 Asn Asn Arg Ala Ser Phe Ser Gln Tyr Gly Ala Gly Leu Asp Ile Val180 185 190 Ala Pro Gly Val Asn Val Gln Ser Thr Tyr Pro Ile Ser Thr TyrAla 195 200 205 Ser Leu Asn Gly Thr Ser Met Ala Thr Pro His Val Ala GlyAla Ala 210 215 220 225 Ala Leu Val Lys Gln Lys Asn Pro Ser Trp Ser AsnVal Gln Ile Arg 230 235 240 Asn His Leu Lys Asn Thr Ala Thr Ser Leu GlySer Thr Asn Leu Tyr 245 250 255 Gly Ser Gly Leu Val Asn Ala Glu Ala AlaThr Arg 260 265

What is claimed is:
 1. A cleaning composition comprising: (a) from about0.1% to about 10% by weight of protease enzyme which is a carbonylhydrolase variant having an amino acid sequence not found in nature,which is derived from a precursor carbonyl hydrolase consisting of asubstitution of a different amino acid for a plurality of amino acidresidues at a position in said precursor carbonyl hydrolase equivalentto position +210 in Bacillus amyloliquefaciens subtilisin, incombination with one or more amino acid residue positions equivalent tothose selected from the group consisting of +33, +62, +67, +76, +100,+101, +103, +104, +107, +128, +129, +130, +132, +135, +156, +158, +164,+166, +167, +170, +209, +215, +217, +218, and +222 in Bacillusamyloliquefaciens subtilisin, provided that: when said carbonylhydrolase variant includes a substitution at positions equivalent to+210 and +76, there is also a substitution of an amino acid residue atone or more of said amino acid residue positions other than amino acidresidue positions equivalent to positions +101, +103, +104, +107, +128,+135, +156, +166, +217, +218 and +222; and (b) one or more cleaningadjunct materials compatible with the protease enzyme.
 2. A cleaningcomposition according to claim 1 of wherein said cleaning composition isa dishwashing detergent composition comprising: (a) from about 5% toabout 90% by weight of the composition of a builder; (b) from about 0.1%to about 15% by weight of the composition of detersive surfactant; (c)optionally, from about 0.1% to about 40% by weight of the composition ofa bleaching agent; and (d) cleaning adjunct materials.
 3. Thecompositions according to claim 1 wherein the cleaning adjunct materialsare selected from the group consisting of surfactants, solvents,buffers, enzymes, soil release agents, clay soil removal agents,dispersing agents, brighteners, suds suppressors, fabric softeners, sudsboosters, enzyme stabilizers, builders, bleaching agents, dyes,perfumes, and mixtures thereof.
 4. The composition according to claim 3further comprising from about 5% to about 50% of a builder selected fromthe group consisting of zeolites, polycarboxylates, layered silicates,phosphates, and mixtures thereof.
 5. The compositions according to claim4 wherein the cleaning adjunct materials comprise at least one bleachingagent.
 6. The compositions according to claim 5 wherein the bleachingagent is selected from the group consisting of percarbonates,perborates, and mixtures thereof, and optionally further comprising atleast one bleach activator.
 7. The compositions according to claim 6wherein the composition further includes at least one detersive enzymeselected from the group consisting of cellulases, lipases, amylases,phospholipases, proteases, peroxidases and mixtures thereof.
 8. A methodfor cleaning fabric, said method comprising contacting a fabric in needof cleaning with the composition as claimed in claim
 1. 9. A method forcleaning dishes, said method comprising contacting a dish in need ofcleaning with a composition according to claim
 1. 10. A method forpersonal cleansing, said method comprising contacting the part of thehuman or lower animal body in need of cleaning with a compositionaccording to claim
 1. 11. A method for providing improved spotting andfilming benefits in automatic dishwashing comprising contacting a dishin need of cleaning with a composition according to claim 1.